1. Field of the Invention
The present invention relates to a multivesicular liposome composition of liposomes encapsulating biologically active substances. More particularly, the present invention relates to methods to produce and use multivesicular liposomes with controllable release rate of the encapsulated substance.
2. Description of Related Art
Optimal treatment with many drugs often requires that the drug level be maintained for a prolonged period of time. For example, optimal anti-cancer treatment with cell cycle-specific antimetabolites requires maintenance of a cytotoxic drug level for a prolonged period of time. Cytarabine is a highly schedule-dependent anti-cancer drug. Because this drug kills cells only when they are synthesizing DNA, prolonged exposure at therapeutic concentration of the drug is required for optimal cell kill. The therapeutic effectiveness of such agents is often further complicated by the fact that the half-life after an intravenous or subcutaneous dose may be as short as a few hours. To achieve optimal cancer cell kill with a cell cycle phase-specific drug like cytarabine, there are two major requirements: first, the cancer cell must be exposed to a high concentration of the drug without doing irreversible significant harm to the host; and second, the tumor must be exposed to the drug for a prolonged period of time to maximize the number of cancer cells which are in the susceptible DNA synthesis cycle of cell proliferation.
An example of another class of drugs that are schedule-dependent is the aminoglycoside class of antibiotics. For instance, amikacin is an aminoglycoside antibiotic which has clinically significant activity against strains of both gram negative and gram positive bacteria. Under existing therapeutic procedures, the drug is normally administered by intravenous or intramuscular routes on a once or twice a day schedule. The most commonly used clinical dose is 15 mg/Kg/day which is equivalent to a maximum recommended daily dose of 1 g per day. However, this approach results in systemic exposure to the patients, and depending on the drug, increases risk of toxic side effects. Consequently, a local depot slow-release preparation for treatment of infections such as those confined to a local region of soft tissue or bone would be advantageous in increasing local tissue levels of the drug, compared with therapeutic systemic doses, while reducing or avoiding the systemic toxicity of the free drug.
Especially useful would be controlled release preparations that can be used to deliver therapeutic levels of drugs over periods ranging from hours, days, or even weeks.
One approach which has been used to provide controlled release compositions for drug delivery is liposome encapsulation. Among the main types of liposomes, multivesicular liposomes (Kim, et al., Biochim. Biophys. Acta; 728:339-348, 1983), are uniquely different from unilamellar liposomes (Huang, Biochemistry; 8:334-352, 1969; Kim, et al., Biochim. Biophys. Acta; 646:1-10, 1981), multilamellar liposomes (Bangham, et al., J. Mol. Bio., 13:238-252, 1965), and stable plurilamellar liposomes (U.S. Pat. No. 4,522,803). In contrast to unilamellar liposomes, multivesicular liposomes contain multiple aqueous chambers. In contrast to multilamellar liposomes, the multiple aqueous chambers of multivesicular liposomes are non-concentric. The prior art describes a number of techniques for producing various types of unilamellar and multilamellar liposomes; for example, U.S. Pat. No. 4,522,803 to Lenk; U.S. Pat. No. 4,310,506 to Baldeschwieler; U.S. Pat. No. 4,235,871 to Papahadjopoulos; U.S. Pat. No. 4,224,179 to Schneider; U.S. Pat. No. 4,078,052 to Papahadjopoulos; U.S. Pat. No. 4,394,372 to Taylor; U.S. Pat. No. 4,308,166 to Marchetti; U.S. Pat. No. 4,485,054 to Mezei; and U.S. Pat. No. 4,508,703 to Redziniak; Szoka, et al., 1980, Ann. Rev. Biophys. Bioeng., 9:465-508; Liposomes, Marc J. Ostro, Ed., Marcel-Dekker, Inc., New York, 1983, Chapter 1; Poznansky and Juliano, Pharmacol. Rev., 36:277-236, 1984. The prior art also describes methods for producing multivesicular liposomes (Kim, et al., Biochim. Biophys. Acta, 728:339-348, 1983).
In the method of Kim, et al., (Biochim. Biophys. Acta, 728:339-348, 1983) the encapsulation efficiency of some small molecules such as cytosine arabinoside, also known as cytarabine or Ara-C, was relatively low, and the release rate of encapsulated molecules in biological fluids was high. Subsequently, a composition was developed which used hydrochloride to slow the release rate of encapsulated molecules in biological fluids (U.S. Patent continuation-in-part application of application Ser. No. 08/020,483, filed Feb. 21, 1993, by Kim).
Further research has shown that the release rate of substances from multivesicular liposomes in human plasma can be controlled by means of varying the nature of the acid solution in which the substance is dissolved prior to forming the multivesicular liposome (U.S. patent application Ser. No. 08/153,657, filed Nov. 16, 1993, Sankaram and Kim). In these studies, certain mineral acids such as hydrochloric, nitric and perchloric acids produced the slowest release rates, whereas other acids, such as acetic, trifluoroacetic, and trichloroacetic resulted in intermediate release rates. The fastest release rates were obtained using di- and tri-protic acids such as sulfuric and phosphoric acids. The composition of liposomes produced by this method has the disadvantage that it requires the use of acid to produce a multivesicular liposome with desirable drug release kinetics. Furthermore, some of the acids which might provide desirable drug release rates from multivesicular liposomes may present pharmaceutical process problems, such as corrosion of metallic containers and parts used in the manufacture. Also, potential problems with the stability of acid-labile substances could be avoided if multivesicular liposomes which are practical in an in vivo environment could be produced without the use of acid.
Studies have shown that the rate of release of the encapsulated biological substance from liposomes into an aqueous environment can be modulated by creating a membrane potential by introducing protonophores or ionophores into liposomes (U.S. Pat. No. 5,077,056). In addition, a method for controlling the release rate of drugs from vesicle compositions is disclosed in European Patent Application EP 0 126 580. In this latter method, a composition was provided comprising a solution of a therapeutic agent encapsulated in vesicles which were suspended in a solution containing sufficient solute to provide a certain osmolarity. This osmolarity was at least substantially isotonic with respect to the osmolarity of the solution within the vesicles which, in turn, had a greater osmolarity than physiological saline. The osmolarity of the solution within the vesicles was held constant, while the osmolarity of the solution in which the vesicles are suspended was varied. Under these conditions, the release rate decreased as the suspending medium approached an isotonic, or even hypertonic relationship with respect to the solution within the vesicles. However, a drawback of this method is that the osmolarity of the medium into which the therapeutic agent is released must be varied in order to control the rate of release. As a consequence, these compositions are severely limited in terms of their practical use, for example, in pharmaceutical applications, since the osmolarity of the biological fluid at the site of administration of the drug delivery system is substantially fixed and cannot be varied. For example, biological fluids such as plasma, are close to isotonic with respect to normal saline (0.9 wt % NaCl in water), which has an osmolarity of 310 mOsm. Therefore, the osmolarity of the suspending medium should ideally be close to 310 mOsm.
In view of the perceived limitations with existing liposome compositions and production methodology, techniques which do not rely upon the use of acids for production of stable multivesicular liposomes are needed. The present invention addresses this need.